Major Changes of von Willebrand Factor Multimer Distribution in Cirrhotic Patients with Stable Disease or Acute Decompensation

Abstract

Background: There is an unstable balance between pro- and anti-haemostatic processes in patients with cirrhosis. We hypothesized, that in patients with acute decompensation (AD) the major alterations of von Willebrand factor (VWF) could contribute to the pro-thrombotic situation as compared to patients with stable (ST) cirrhosis.

Patients and methods: We analysed different parameters of VWF, including detailed multimer distribution by densitometry and platelet adhesion, together with adisintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) activity and antigen and C-reactive protein (CRP) levels in patients with ST cirrhosis (n = 99), with AD (n = 54) and controls (n = 92).

Results: VWF antigen, ristocetin co-factor as well as collagen-binding activities were elevated in both cirrhotic groups in a stepwise manner. There was a decrease in high and an increase in low molecular weight multimer ratios in the majority of ST cirrhosis. However, in 24 out of 54 AD patients, ultra-large VWF multimers (ultra-large molecular weight multimers [ULMWM]) were found. ADAMTS13 activity in ST and AD patients without ULMWM was similar to controls (median [interquartile range; IQR]%: 98 [67-132] and 91 [60-110] vs. 106 [88-117], respectively). The presence of ULMWM in AD patients was associated with low ADAMTS13 activity [33 (24-49)%] and high CRP level [23 (7.1-83.6) mg/L]. Adhesion of normal platelets showed a stepwise increase in the presence of cirrhotic plasmas, reaching the highest level in AD patients with ULMWM.

Conclusion: Characteristic changes of VWF parameters are seen in ST cirrhosis. In AD patients, highly increased VWF and reduced ADAMTS13 activity could be found, along with the presence of ULMWM, which are possible markers and contributors of the disease progression.

Fig. 1

Plasma von Willebrand factor antigen (VWF:Ag), pro-peptide (VWFpp) and VWF functional activities in cirrhosis. VWF:Ag ( A ), VWF ristocetin co-factor activity (VWF:RCo) ( B ), VWF collagen-binding activity (VWF:CB) ( C ),VWFpp ( D ), VWF:RCo/VWF:Ag ( E ) and VWF:CB/VWF:Ag ( F ) in plasma of healthy controls and patients with stable (ST) cirrhosis and acute decompensation (AD) are shown. VWF:Ag, VWFpp and VWF activity levels are expressed as percentage of pooled normal plasma. Lines denote median values, boxes represent 25th to 75th percentiles and whiskers indicate the 5th to 95th range. p -Values were calculated by Kruskal–Wallis H test with Dunn's multiple comparison post hoc analysis.

Fig. 2

Multimeric pattern of von Willebrand factor (VWF) after separation on 0.8% agarose gel and the corresponding densitometric curves. ( A ) Characteristic plasma sample of a patient with acute decompensation (AD), stable (ST) cirrhosis and healthy control. The presence of ultra-large molecular weight multimers (ULMWM) is seen in the sample of the AD patient. No ULMWM can be seen in the sample of ST cirrhosis or healthy control. The dotted lines across the lanes represent critical separation on the distribution ladder according to the number of the corresponding bands. The critical separation lines: #1 = 1 dimer; #6 = 6 dimers and #10 = 10 dimers. Dimers < 6 are defined as low molecular weight multimers (LMWM), dimers between 6 and 10 defined as intermediate molecular weight multimers (IMWM) and dimers > 10 defined as high molecular weight multimers (HMWM) including ULMWM. ( B ) The corresponding densitometric curves indicated with different lines and areas as marked at the bottom of A .

Fig. 3

Proportions of the low molecular weight multimers (LMWM), intermediate molecular weight multimers (IMWM) and high molecular weight multimers (HMWM) groups of bands and the corresponding absolute amount of von Willebrand factor (VWF) in each group of multimers. ( A ) The proportion of the area under the curve (AUC) of the first 5 bands considered as LMWM (1–5 dimers). ( B ) The proportion of the AUC of bands between 6 and 10 named as IMWM (6–10 dimers). ( C ) The proportion of the AUC of larger than 10 multimers, considered as HMWM including the area where VWF did not resolve into bands anymore (> 10 dimers, HMWM). ( D – F ) The absolute amount of VWF within the LMWM, IMWM and HMWM groups of multimers. The group of stable (ST) and acute decompensation (AD) patients are separated according to the presence or absence of ultra-large molecular weight multimers (ULMWM). Lines denote median values, boxes represent 25th to 75th percentiles and whiskers indicate the 5th to 95th range. p -Values were calculated by Kruskal–Wallis H test with Dunn's multiple comparison post hoc analysis.

Fig. 4

Plasma a d isintegrin-like a nd m etalloproteinase with t hrombo s pondin type-1 motifs 13 (ADAMTS13) activity ( A ) and antigen level ( B ) in plasma of healthy controls and patients with stable (ST) cirrhosis and acute decompensation (AD) separated according to the presence of ultra-large molecular weight multimers (ULMWM). ADAMTS13 activity and antigen levels are expressed as percentage of pooled normal plasma. Lines denote median values, boxes represent 25th to 75th percentiles and whiskers indicate the 5th to 95th range. p -Values were calculated by Kruskal–Wallis H test with Dunn's multiple comparison post hoc analysis.

Fig. 5

Linear regression line and correlation between the quantity of von Willebrand factor (VWF) high molecular weight multimers (HMWM) and a d isintegrin-like a nd m etalloproteinase with t hrombo s pondin type-1 motifs 13 (ADAMTS13) activity. HMWM VWF antigen (VWF:Ag) levels were plotted versus the plasma ADAMTS13 activities of healthy controls ( A ), Spearman's r  = –0.043, p  = 0.722, n  = 70; stable cirrhosis patients ( B ), Spearman's r  = –0.18, p  = 0.073, n  = 98; and patients with acute decompensation ( C ), Spearman's r  = –0.43, p  = 0.01, n  = 54.

Fig. 6

Prothrombotic tendency in patients with stable (ST) cirrhosis and acute decompensation (AD). ( A ) Adhesion of normal platelets in the presence of plasma of healthy controls or patients. Platelet adhesion activity of plasma from patients with cirrhosis was studied using reconstituted blood at 1,800/s, 2.5 minutes using a cone and plate analyser with uncoated wells (DiaMed Impact-R). Ten ST, 10 AD patients and 10 controls were selected. The number of platelets (Plt) adhering were determined by acid phosphatase activity. Results of cirrhotic samples are separated according to the absence or presence of ultra-large molecular weight multimers (ULMWM). Lines denote median values. p -Values were calculated by Mann–Whitney and Kruskal–Wallis H tests. ( B ) Thrombin generation in plasma of controls and both cirrhotic patient groups. Samples from 31 controls, 62 ST and 27 AD cirrhotic patients were analysed using the Technothrombin kit and results are expressed as peak thrombin concentration. Lines denote median values, boxes represent 25th to 75th percentiles and whiskers indicate the 5th to 95th range. p -Values were calculated by Kruskal–Wallis H test with Dunn's multiple comparison post hoc analysis.

Fig. 7

Survival of cirrhotic patients. Receiver operating characteristics curves for prediction of 6-month survival for von Willebrand factor antigen (VWF:Ag) (dotted line), VWF collagen-binding activity (VWF:CB) (solid line) and a d isintegrin-like a nd m etalloproteinase with t hrombo s pondin type-1 motifs 13 (ADAMTS13) activity (dashed line), indicating the best nominal area under the curve (AUC) for VWF:CB ( A ). Kaplan–Meier curves indicate higher 6-month survival for patients with < 245% VWF:CB ( B ), or with > 50% ADAMTS13 activity ( C ) or < 10 mg/L C-reactive protein (CRP) level ( D ).